1. Field of the Invention (Technical Field)
This invention relates to a method and composition for radiolabeling monoclonal antibodies against stage specific embryonic antigen-1 with the radioisotope technetium-99m, and further providing for use of the radiolabeled monoclonal antibody preparation for detection of infections and inflammation.
2. Background Art
The use of radioisotopes to label proteins is well known. These compositions can be used in in vitro assays; can be administered to the human body to visualize or monitor functioning of various parts of the body or to determine the presence and location of particular antigens, antibodies, hormones or the like; and can be used in the treatment of various disease states.
Technetium-99m has been utilized to radiolabel proteins, chelating agents, phosphonate bone scanning compositions and the like by a technique which utilizes sodium pertechnetate wherein the technetium initially is in the +7 state. Technetium-99m is generally available only as sodium pertechnetate. The pertechnetate comes into contact with a reducing agent, such as stannous chloride, for the technetium to be reduced to the +3, +4 or +5 oxidation state. The technetium must be maintained in this reduced state in order to maintain the chemical bond between the technetium molecule and the protein, chelating agent or like substrate being radiolabeled. It is also necessary that the technetiumbe firmly bound to the protein such that the reduced technetium is not transferred to other molecules or other proteins present in the assay, patient's blood or other media in which the radiolabeled substance will be utilized.
Several different methods have been utilized to radiolabel proteins, particularly monoclonal antibodies, with technetium-99m. The methods involve two general approaches. One approach is indirect in which a bifunctional chelating agent is attached to the protein via one functional group and the technetium-99m is attached via the other functional, or chelating, group. This method was introduced by Krejcarek, G. E. and Tucker, K. L. ("Covalent Attachment of Chelating Groups to Macromolecules," Biochemical and Biophysical Research Communications 77:581-585, 1977) and has been widely employed in many variations using a wide variety of bifunctional chelating agents such as described in the review of Wensel and Meares (Wensel, T. G. and Meares, C. F., "`Bifunctional` Chelating Agents for Binding Metal Ions to Proteins," Radioimmunoimaging and Radioimmunotherapy, S. W. Burchiel and B. A. Rhodes, eds., Elsevier Publishing Co., New York, 1983, pp 185-196). Other methods are disclosed by Hnatowich, D. J., U.S. Pat. Nos. 4,668,503 and 4,479,930; by Meares, C. F. et. al., U.S. Pat. No. 4,622,420; by Haber, E., and Khaw, B. A., U.S. Pat. No. 4,421,735; and by Fritzberg, A. R., and Kastna, S., U.S. Pat. No. 4,670,545, and by Baidoo, K. E., et. al., ".sup.99m Tc Labeling of Proteins: Initial Evaluation of Novel Dtamtnedithiol Bifunctional Chelating Agent," Cancer Res (Supp) 50:799s-803s, 1990. The bifunctional chelate methods all present significant limitations, including the complexity of the radiolabeling procedure, the time required to accomplish radiolabeling, and the presence of substances which may affect the protein.
The other general approach is direct labeling. Although several direct methods have been reported, the first direct method capable of providing a sufficiently strong bond between the protein and the technetium-99m for in vivo applications was the direct or pretinning method described in U.S. Pat. No. 4,424,200, entitled Method for Radiolabeling Proteins with Technetium-99m, to Crockford, D. R., and Rhodes, B. A. In this method, a single reduction compound, consisting of stannous [Sn(II)] chloride and other salts which serves both to reduce the protein, thereby exposing the disulfide bonds, and to reduce the sodium pertechnetate, is used. With this method, many proteins can be successfully radiolabeled with .sup.99m Tc. Several investigators have reported on the use of this method (Rhodes, B. A., et. al., "Technetium-99m labeling of murine monoclonal antibody fragments," J Nucl Med 27:685-693, 1986; Som, P., et. al., "Radioimmunoimaging of experimental thrombi in dogs using technetium-99m-labeled monoclonal antibody fragments reactive with human platelets," J Nucl Med 27:1315-1320, 1987).
Equivalent methods for direct labeling have been reported (Schwarz, A., and Steinstruaber, A., "A novel approach to Tc-99m-labeled monoclonal antibodies," J Nucl Med 28:721, 1987; Pak, K. Y., et. al., "A rapid and efficient method for labeling IgG antibodies with Tc-99m and comparison to Tc-99m Fab'". J Nucl Med 30:793, 1989; Granowska, M., et. al., "A Tc-99m-labeled monoclonal antibody, PR1A3, for radioimmunoscintigraphy," J Nucl Med 30:748, 1989). In the equivalent methods disulfide reducing agents other than stannous salts were used. Pak et. al. used dithiothreitol to reduce the disulfide bonds of the antibody; Swartz and Steinsbruaber, and Granowska et. al. used 2-mercaptoethanol. Also some of these investigators (Swartz and Stetnsbruaber, and Granowska et. al.) reduced the Tc-99m prior to adding it to the reduced antibody, which adds steps to the original procedure.
Reno, J. W., et. al., U.S. Pat. No. 4,877,868, Radionuclide Antibody Coupling, uses dithiothreitol (DTT) to reduce the disulfide groups of the protein, then protect the reactive sulfides with Zn (II) or other sulfhydryl group dertvatiztng reagents. Tartrate salts are used to complex and transfer the reduced radionuclide. This method uses potentially toxic chemicals, such as dithiothreitol, to reduce the antibody. It also requires multiple steps to radiolabel the protein.
Thakur, M. L., U.S. Pat. No. 5,011,676, Method to Directly Radiolabel Antibodies for Diagnostic Imaging and Therapy, used sodium ascorbate to reduce the disulfide groups of antibodies. This method was used with anti-SSEA-1 IgMneutrophil specific antibody. However, this method cannot be adapted to single-step, direct labeling; it is required to reduce the radionuclide prior to adding the radionuclide to the sodium ascorbate reduced protein. In a preferred embodiment of the Thakur method, a separate vial is utilized, in which sodium dithionite is used to reduce the radionuclide, producing dithionite reduced radionuclide, and the reduced radionuclide is then added to the sodium ascorbate reduced protein.
In U.S. Pat. No. 4,917,878, Novel Use of a Radiolabelled Antibody Against Stage Specific Embryonic Antigen for the Detection of Occult Abscesses in Mammals to M. L. Thakur, a class of antibody, useful when radiolabeled for the diagnosis of hidden infections and inflammations in man, is identified. This class of antibody comprises antibody against stage specific embryonic antigen-1 (hereinafter "SSEA-1" or "anti-SSEA-1"). The SSEA-1 antigen is known to be expressed on human granulocytes. Anti-SSEA-1 antibodies have high specificity for human neutrophils, and thus preferentially bind to human neutrophils in vivo.